Parvalbumin-expressing basal forebrain neurons mediate learning from negative experience

Parvalbumin (PV)-expressing GABAergic neurons of the basal forebrain (BFPVNs) were proposed to serve as a rapid and transient arousal system, yet their exact role in awake behaviors remains unclear. We performed bulk calcium measurements and electrophysiology with optogenetic tagging from the horizontal limb of the diagonal band of Broca (HDB) while male mice were performing an associative learning task. BFPVNs responded with a distinctive, phasic activation to punishment, but showed slower and delayed responses to reward and outcome-predicting stimuli. Optogenetic inhibition during punishment impaired the formation of cue-outcome associations, suggesting a causal role of BFPVNs in associative learning. BFPVNs received strong inputs from the hypothalamus, the septal complex and the median raphe region, while they synapsed on diverse cell types in key limbic structures, where they broadcasted information about aversive stimuli. We propose that the arousing effect of BFPVNs is recruited by aversive stimuli to serve crucial associative learning functions.


Supplementary Figures
Average spike waveform of the same BFPVN on the four tetrode channels (blue, average lightevoked spikes; black, average spontaneous spikes; grey, all spikes).f, Spike clusters plotted in feature space from an example recording session.g, Average spike waveforms of the recorded neurons on each tetrode channel from the same session.Table S3.Secondary antibodies used in immunohistochemical experiments.
Figure S1.Optogenetic tagging of HDB BFPVNs.a, Cumulative histogram of the peak response latency of BFPVNs after optogenetic stimulation (n = 36).b, Cumulative histogram of the jitter of BFPVN spike responses after optogenetic stimulation (n = 36).c, Distribution of the significance values of the SALT statistical test (H-index) for all recorded neurons (blue, p < 0.01, tagged BFPVNs; grey, p > 0.01, untagged neurons).d, Example spike raster and PETH of an optogenetically tagged BFPVN responding to 20 Hz blue laser light stimulation.e,

Figure S2 .
Figure S2.HDB BFPVNs are not modulated by outcome expectation.a, Left, average PETH of BFPVNs aligned to cue onset (n = 36).Right, difference of peak response to Cue 1 and Cue 2 (in a 0-0.5s time window from cue onset).n.s., p > 0.05, p = 0.6151, two-sided Wilcoxon signed-rank test.b, Left, average PETH of BFPVNs aligned to expected and surprising reward (n = 36).Right, difference of peak response to expected and surprising reward.n.s., p > 0.05, p = 0.8628, two-sided Wilcoxon signed-rank test.c, Left, average PETH of BFPVNs aligned to expected and surprising punishment (n = 29, 7 neurons excluded from this analysis because there were only 5 or less surprising punishments in the session).Right, difference of peak response to expected and surprising punishment.n.s., p > 0.05, p = 0.5566, two-sided Wilcoxon signed-rank test.Source data are provided as a Source Data file.

Figure
Figure S3.K-means clustering of BF neuronal responses reveals groups of neurons with distinct firing patterns.a, Color-coded, Z-scored PETHs of all neurons aligned to Cue 1 (left) and Cue 2 (right; n = 685).Red asterisks indicate tagged BFPVNs.The clusters were ordered according to percentage of tagged neurons.b, Top and middle, PETH of example neurons aligned to stimulus (top) and reinforcement (middle).Bottom, Average, Z-scored PETH of each cluster.

Figure S5 .
Figure S5.Electrophysiological properties of HDB BFPVNs.a, Average PETH of burst spikes and single spikes aligned to punishment onset (n = 36; errorshade, SEM).b, BFPVNs were partitioned based on burst index and refractory period.Neurons with high (> 0.3) burst index and short (< 2 ms) refractory period were considered as bursting neurons.Neurons with low burst index and/or long refractory period were considered non-bursting.c, Average autocorrelogram of bursting (left) and non-bursting (right) neurons (errorshade, SEM).d, Spike shape features of BFPVNs (peak-to-post-valley time and post-valley magnitude, normalized to the integral).Most bursting neurons had smaller valley and longer peak-to-post-valley time.e, Average spike shape of bursting and non-bursting neurons (errorshade, SEM).f, Correlation of burst index and peak-to-post-valley time.g, Average PETH of bursting BFPVNs aligned to punishment (errorshade, SEM).Pie chart showing activation, suppression or no response to

Fig S8 .
Fig S8.Track reconstruction and isosbestic channels of fiber photometry recordings.a, Representative fluoromicrograph of a GCaMP6s injection in a PV-Cre mouse (repeated in n = 12 mice).b, Reconstruction of all injection sites in the HDB.c, PETH of 465 nm and 405 nm wavelength fluorescent signals aligned to punishment, recorded in the HDB (n = 19 sessions;